ITAM-mediated Signaling by the T-Cell Antigen...

ITAM-mediated Signaling by the T-CellAntigen Receptor

Paul E. Love1 and Sandra M. Hayes2

1Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes ofHealth, Bethesda, Maryland 20892

2Department of Microbiology & Immunology, SUNY Upstate Medical University, Syracuse, New York 13210

Correspondence: [email protected]

Signal transduction by the T-cell antigen receptor (TCR) is initiated by phosphorylationof conserved motifs (ITAMs) contained within the cytoplasmic domains of the invariantsubunits. TCR complexes contain a total of 10 ITAMs and this unusual configurationhas prompted studies of the role of specific ITAMs, or of ITAM multiplicity, in regulatingTCR-directed developmental and effector responses. Here, we summarize data generatedduring the past two decades and discuss how these findings have in some cases resolved,and in others complicated, outstanding questions relating to the function of TCR ITAMs.

Signal transduction in the immune systemis regulated by a highly diverse set of cell

surface receptors that are coupled by signalingintermediates to common downstream path-ways. A large number of these receptors eithercontain within their cytoplasmic domains,or associate with subunits that contain, a con-served sequence (the Immune-receptor-Tyro-sine-based-Activation-Motif; ITAM) that iscritical for the initiation of signaling followingligand engagement (reviewed in Underhill andGoodridge 2007). ITAMs, which were identified20 years ago on the basis of their sequencehomology (Reth 1989), consist of paired YxxL/I motifs separated by a defined interval (YxxL/I-X6-8-YXXL/I). In addition, most ITAMs con-tain a negatively charged amino acid (D/E) inthe þ2 position relative to the first ITAM

tyrosine (Fig. 1). ITAM containing receptorsare widely expressed in hematopoietic cellsincluding T and B lymphocytes, naturalkiller (NK) cells, macrophages, dendritic cells,and platelets (Fig. 2). Since their discovery,considerable progress has been made in deter-mining how ITAMs initiate signaling cascadesand thereby couple their associated receptorsto distal effector responses. In this review,we focus on the TCR as a paradigm for ITAM-mediated signal initiation. In addition tobeing one of the most well characterized recep-tor complexes, the TCR exhibits several in-triguing properties, such as the inclusion of anespecially large number of ITAM containingsubunits, which have fueled intensive studyinto the role of ITAMs in the TCR signalingresponse.

Editors: Lawrence E. Samelson and Andrey Shaw

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ITAMS WITHIN THE TCR SUBUNITS

TCRs are multimeric complexes composed ofsubunits whose function is exclusively limitedto either ligand recognition or signal trans-duction. The predominant TCR isoform, theabTCR, which is expressed on mature CD4þ

and CD8þ T cells, contains clonotypic TCRaand TCRb chains that form a heterodimer con-ferring peptide-major histocompatibility com-plex (MHC) binding specificity to the TCR.The TCRa/b heterodimer lacks inherent signaltransducing activity but associates noncova-lently with multiple signal transducing sub-units: the CD3g,-d and -1 chains, and, in most

cases, a z chain homodimer. The currentlyaccepted model of abTCR stoichiometry pro-poses the following subunit composition:TCRab, CD3g1, CD3d1, zz. A distinct lineageof T cells (gd T cells) expresses a TCR complexcontaining a different antigen recognition het-erodimer composed of chains encoded by theTCRg and TCRd genes. Although the invariantsubunit composition of the abTCR and thegdTCR had long been assumed to be identical,it was recently shown that the gdTCR lacksCD3d (Hayes and Love 2002), and thereforemost likely has the following stoichiometry:TCRgd, CD3g1, CD3g1, zz (Hayes and Love2006a; Siegers et al. 2007). A third complex,the pre-TCR, is expressed only during early thy-mocyte development at a stage that precedesrearrangement of the TCRa gene. In lieu ofTCRa, TCRb associates with an invariantchain (pre-Ta) encoded by a gene that doesnot undergo V-(D)-J rearrangement (Saint-Rufet al. 1994). Although the subunit compositionof the pre-TCR has not been unequivocallyestablished, the phenotype of knockout micelacking individual TCR subunits suggests thatit likely contains at least CD3g1 and zz dimersin addition to the TCRb and pre-Ta chains(Hayes et al. 2003).

A striking feature of the TCR (and pre-TCR)structures is the presence of multiple distinctITAM bearing subunits within the receptorcomplex. The CD3 (2g, 2d, 21) chains eachcontain a single ITAM whereas the z chain

CD3ε CD3δ CD3γ

Igα Igβ

TCRζ-1 TCRζ-2 TCRζ-3

FcεRIβ FcεRIγ

DAP12

Consensus

D--YEPIRKGQRDL–YSGLDQLYQPLRDREDTQ–YSRLEQLYQPLKDREYDQ–YSHL

ENLYEGLNLDDCSM–YEDIDHTYEGLDIDQTAT–YEDI

NQLYNELNLGRREE–YDVL

EGVYNALQKDKMAEAYSEIDGLYQGLSTATKDT–YDAL

DRVYEELNIYSAT--YSELDGVYTGLSRNQET--YETL

ESPYQELQGQRSDV–YSDL

Figure 1. ITAM-containing proteins. Murine ITAMsequences were obtained from GenBank. Aminoacids that align with the consensus ITAM sequenceare shown in blue.

CD3δε CD3γε

DAP12-DAP12

lgα/lgβ

TCRαβ

TCR BCR FCεRI LY49

LY49

mlg

β

α

γ-γζ-ζ

Figure 2. Comparison of the ITAM-containing T- and B-cell antigen receptors, Fc receptors and NK receptors.Subunit composition of the T-cell antigen receptor (TCR), B-cell antigen receptor (BCR), Fc1R1 (an example ofan activating Fc receptor) and Ly49 (an example of an activating NK receptor). Blue rectangles represent ITAMs.

P.E. Love and S.M. Hayes

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contains three tandem ITAMs (Fig. 1 andFig. 2). Consequently, the predicted octamericabTCR and gdTCR complexes contain a totalof 10 ITAMs. Moreover, although all TCRITAMs share the conserved YxxL/I-X6-8-YXXL/I motif, the amino acid sequence of eachITAM is distinctive (Fig. 1). The question thatnaturally arises is whether the individual TCRITAMs have specific signaling responses duringT-cell development and T-cell activation or in-stead perform equivalent but additive functions.

INITIATION OF TCR SIGNALING BY ITAMS

The essential role of ITAMs in the initiation ofTCR signaling was first established by experi-ments demonstrating that the cytoplasmicdomains of the CD3 or z chains, or the individ-ual ITAM sequences from these proteins, werecapable of activating T cells when fused tothe extracellular domain of unrelated mole-cules (Irving and Weiss 1991; Letourneur andKlausner 1992; Romeo et al. 1992; Straus andWeiss 1993; Koyasu et al. 1994; Sturmhofelet al. 1995). It had been shown previously that

tyrosine phosphorylation of TCR subunits,which represents one of the earliest eventsfollowing TCR engagement, was mediated bythe Src family tyrosine kinases Lck and Fyn(Samelson et al. 1986; June et al. 1990; Samelsonet al. 1990) and that Lck associates con-stitutively with the CD4 and CD8 coreceptors(Veillette et al. 1988; Barber et al. 1989). Animportant breakthrough was the subsequentidentification of ZAP-70, a Syk family tyrosinekinase that is rapidly recruited to phosphory-lated ITAMs (Chan et al. 1992). Both ZAP-70and Syk contain tandem SH2 domains thatdirect their specific and selective interactionwith doubly phosphorylated ITAMs. The com-binatorial action of Src kinases and ZAP-70 issufficient for the full activation of downstreamsignaling pathways and for eliciting T-cell effec-tor responses (Fig. 3).

A series of experiments performed over thepast few years have served to clarify and refinethe proximal events that take place followingTCR engagement. A fundamental question hasbeen how ligand binding promotes ITAM phos-phorylation and thereby leads to the initiation

TCR

LAT

CD4

Actincytoskeletal

rearrangement

RacCdc42

Raf

Ras

Mek

Erk

Phosphorylation of transcription

factors NF-AT

DAGIP3

Ca++

Calcineurin

JNK/p38?

PKC

P

ZAPPP

PLC-γ

Grb2ItKP

GadsSos

RasGRP

Lck

SLP-76

NckVavP P

P

P

P

Figure 3. Proximal signaling events in the TCR-coupled signaling pathway. Interactions between the TCR andpeptide-MHC results in the activation of Lck, a member of the Src family of tyrosine kinases. Lck thenphosphorylates the two-tyrosine residues within the ITAMs of the CD3 and z chains. A second tyrosinekinase, ZAP-70, is specifically recruited to biphosphorylated ITAMs. Phosphorylation of ZAP-70 by Lckresults in its activation. ZAP-70 and Lck phosphorylate and activate several downstream target proteinseventually leading to Ras activation, calcium mobilization, and actin cytoskeleton rearrangements, andultimately to the activation of transcription factors.

ITAM-mediated Signaling by the T-Cell Antigen Receptor

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of TCR signaling. One explanation, relevant tocells expressing the abTCR, is that cobindingof the peptide-MHC complex by the TCR andby CD4 or CD8 results in the juxtaposition ofLck and the cytoplasmic tails of the invariantTCR subunits resulting in the phosphorylationof ITAMs by Lck (Chu and Littman 1994).However, this mechanism cannot explain howITAMs within the pre-TCR and gdTCR arephosphorylated as these receptors are expressedon cells that do not express either CD4 or CD8.This paradox has led to the proposal of severalnew models [reviewed in (Smith-Garvin et al.2009)] that invoke mechanisms includingligand-induced conformational changes in theTCR, receptor re-distribution on the cell sur-face, or subunit movement relative to the plasmamembrane to explain how TCR engagementcould render ITAMs more accessible to phos-phorylation. One especially attractive modelstems from the observation that the cytoplasmictails of CD31 and z interact with acidic phos-pholipids that are enriched in the inner leafletof the plasma membrane (Aivazian and Stern2000; Sigalov et al. 2006; Xu et al. 2008). Suchinteractions, which would be predicted to resultin the insertion of ITAM tyrosines into thehydrophobic core of the lipid bilayer, couldexplain the inaccessibility of ITAMs as targetsfor phosphorylation in resting T cells. Accord-ing to this model, ligand induced deformationof the TCR leads to dissociation of the CD3and zz dimers from the plasma membrane ren-dering them accessible to phosphorylation byLck and/or Fyn.

The binding of ZAP-70 to phosphorylatedITAMs and its subsequent activation has beenclarified by experiments that systematicallyevaluated the importance of key domains andamino-acid residues within ZAP-70 (reviewedin Au-Yeung et al. 2009). Specifically, thesestudies have shown that ITAM binding byZAP-70 relieves an auto-inhibitory conforma-tion of the kinase and that phosphorylationof specific tyrosine residues within the inter-domain separating the C-terminal SH2 domainand the kinase domain (interdomain B) by Lck/Fyn are important for regulating the activity ofZAP-70 (Au-Yeung et al. 2009).

FUNCTION OF INDIVIDUAL TCR CHAINSAND ITAMS

The discovery that the TCR complex containsseveral different ITAM-bearing subunits imme-diately prompted a series of studies aimed atdetermining whether individual ITAMs wereimportant for particular signaling or effectorresponses. Experiments performed in T-celllines indicated that the cytoplasmic domainsof the CD31 and z subunits were independentlycapable of providing activating signals similarto that of the intact TCR complex (Irving andWeiss 1991; Letourneur and Klausner 1992;Wegener et al. 1992) and suggested that TCRITAMs are functionally redundant but additivewith respect to signaling responses (Irvinget al. 1993). In other studies, the binding ofpotential effector proteins including ZAP-70,Shc, PI3K(p85), Grb2, Fyn, and Ras-GAP tothe TCR ITAMs was analyzed using syntheticphosphorylated peptides containing individualITAM sequences (Isakov et al. 1995; Osmanet al. 1996; Zenner et al. 1996). Only one ofthe three studies compared binding to all 6TCR ITAM sequences (z1, z2, z3, CD32g,2d, and 21) (Osman et al. 1996). Data fromthese experiments provided evidence for pref-erential binding of effector molecules to indi-vidual TCR ITAMs, and in some cases (Shc,p85, Grb2, and Fyn) selective binding to a sub-set of TCR ITAMs (Table 1); however, additionalexperiments to follow up on these initial obser-vations have not be performed. It also remainsunclear if any exclusive ITAM-effector associa-tions can be inferred from these studies becausein vitro analysis of binding to isolated ITAMsmay not reflect binding in the context of theintact TCR where cooperative interactions orordered binding of effectors may take place.

The notion that the extent of TCR ITAMphosphorylation, or the order in which theindividual TCR ITAMs are phosphorylated afterligand engagement, may play a role in regulat-ing the TCR signaling response also formedthe basis of intensive research over the past de-cade. Early experiments revealed that two pro-minent tyrosine phosphorylated forms ofz-chain, p21-z, and p23-z, are detected in T cells





(reviewed in Pitcher et al. 2003b). It was sub-sequently shown that p21-z, which is de-tected in unstimulated ex vivo thymocytes andT cells or in cells activated with partial agonist/antagonist ligands, contains partially phos-phorylated ITAMs that are either not associatedwith ZAP-70 or that bind inactivated ZAP-70,whereas p23-z, which is generated only afterTCR cross-linking or stimulation with agonistpeptides, contains fully phosphorylated ITAMsthat bind activated ZAP-70 (Sloan-Lancasteret al. 1994; van Oers et al. 1994; Madrenaset al. 1995). These findings led to the suggestionthat partial phosphorylation of z-chain may beimportant for the induction of T-cell anergyand may in fact be inhibitory (Kersh et al.1999). One mechanism proposed to explainITAM-mediated inhibition is that partial phos-phorylation of individual ITAMs could resultin the generation of an “ITIM-like” sequence(YXXL/I/V) which then leads to the recruitmentof tyrosine phosphatases including SHP-1 andSHIP to the TCR (Barrow and Trowsdale2006; Pinheiro da Silva et al. 2008). However,other studies implied that p21-z may insteadrepresent a “primed” state that facilitates rapidT-cell activation and that formation of p21-z

through self-MHC interactions may be impor-tant for T-cell survival (Witherden et al. 2000).To address these questions, van Oers and col-leagues engineered transgenic mouse lines thatare capable of generating p21-z but not p23-z,or that are incapable of generating either p21-zor p23-z (Pitcher et al. 2003a). Analysis of thesemice demonstrated that the ability to generatep21-z was not essential for T-cell antagonism.In addition, they found that the ability to gener-ate p23-z was not required for agonist-inducedT-cell proliferation (Pitcher et al. 2003a). Simi-lar results were obtained by other investigatorsusing an identical but independently con-structed experimental system (Ardouin et al.1999). Whether these results indicate that parti-ally phosphorylated TCR ITAMs (or partialphosphorylation of individual TCR ITAMs)have no inhibitory function remains unclear asthe potential for redundancy with other ITAMcontaining subunits still exists in this modelsystem.

To evaluate the importance of TCR signal-ing for T-cell development, in vivo studies wereperformed with gene targeted mice in whichspecific ITAM containing subunits were mu-tated. In an initial series of experiments, mice

Table 1. Differences in the binding affinities of major signaling molecules in the TCR-coupled signaling pathwayfor the ITAMs within the invariant TCR subunits

Signaling Molecule ITAM Binding Hierarchy References

ZAP-70 z1 � z2 . 1 � z3 Isakov et al. 1995a

z1 ¼ g ¼ d . z3 . z2 ¼ 1 Osman et al. 1996b

z1 . z3 . z2 Zenner et al. 1996c

Shc g ¼ d . z3 ¼ z1 Osman et al. 1996

z1. z3 Zenner et al. 1996

p85 regulatory subunit of PI3K z3 ¼ g ¼ d.z1 ¼ z2 Osman et al. 1996

z1 � z2 . z3 Zenner et al. 1996

Grb2 z1 ¼ g ¼ d Osman et al. 1996

z2 Zenner et al. 1996

Fyn z2 ¼ g ¼ d . z1 Osman et al. 1996

Ras-GTPase activating protein (GAP) z2 � z1 . z3 Zenner et al. 1996

aAll 3 TCRz ITAMs and the CD31 ITAM were tested in this study.bAll 6 ITAMs (z1, z2, z3, 1, d, and g) were tested in this study. Only the specific ITAMs that physically interacted with the

signaling molecule are listed.cAll 3 TCRz ITAMs were tested in this study. Only the specific z ITAMs that physically interacted with the signaling molecule

are listed.





lacking expression of the CD3g, 2d, or 21

chains (Malissen et al. 1995; Dave et al. 1997;DeJarnette et al. 1998; Haks et al. 1998) or thez chain (Liu et al. 1993; Love et al. 1993; Malis-sen et al. 1993; Ohno et al. 1993) were generated.Analysis of T-cell development in these micerevealed an important function for CD3g,CD31, and z in regulating the transition of thy-mocytes from the CD42CD82 (Double Nega-tive, DN) to the CD4þCD8þ (Double Positive,DP) stage, an event known to be controlled bypre-TCR signaling (von Boehmer 2005). More-over, each of the invariant TCR subunits wasfound to be required for efficient transitionfrom the DP to the mature CD4þCD82 orCD42CD8þ (Single Positive, SP) stage, whichis dependent upon abTCR signaling (Sebzdaet al. 1999; Starr et al. 2003). Importantly,abTCR surface expression was found to be ad-versely affected in each of the knockout mouselines, results that were in agreement with in vitrostudies showing that all of the TCR subunitsare required for proper assembly and surfaceexpression of the TCR (Klausner et al. 1990).Although these studies clearly demonstrateda critical role for CD3g, 2d, 21, and z inT-cell development, the effects of the gene dele-tions on TCR surface expression meant thatknockout mice could not be used to directlyevaluate the importance of ITAMs and ITAM-mediated signaling.

To specifically address the role of TCRITAMs in thymocyte development, three groupsreconstituted TCRz knockout mice with trans-genes encoding wild-type or mutant forms ofz chain lacking one or more ITAMs, either bydeleting individual ITAMs (Shores et al. 1994)or by mutation of ITAM tyrosines to phenylala-nine (Ardouin et al. 1999; Pitcher et al. 2005b)(Table 2). Significantly, all three studies foundthat none of the TCRz ITAMs was specificallyrequired for thymocyte maturation. However,each study noted that the efficiency of thymocytedevelopment was decreased in the absence ofone or more z ITAMs, and the severity of thedevelopmental block correlated with the numberof inactivated ITAMs but not with the loss of anyspecific ITAM. A non-essential requirement wasalso demonstrated for the CD3g ITAM (Haks

et al. 2002) and CD31 ITAM (Sommers et al.2000) (Table 2). Together, these results stronglysuggested that TCR ITAMs function collectivelyto amplify TCR signals (Love and Shores 2000).

POTENTIAL FUNCTIONS FORITAM-MEDIATED SIGNAL AMPLIFICATIONDURING T-CELL DEVELOPMENT

Recent data indicate that ITAM-mediated signalamplification may have an important role inregulating ab/gd T lineage choice during thy-mocyte development. These experiments werebased on two observations: 1) that ab and gd

T cells originate from a common DN thymocyteprogenitor prior to rearrangement of the TCRalocus (Petrie et al. 1992), and 2) that direct com-parison of the signaling responses of the gdTCRand the abTCR revealed that under equivalentstimulation conditions, the gdTCR delivers astronger signal than the abTCR (Hayes andLove 2002). Since the pre-TCR is expressed atmuch lower levels than the gdTCR on DN thy-mocytes (Hayes and Love 2006b), this raisedthe possibility that the relatively strong signalstransmitted by the gdTCR promote commit-ment to the gd lineage whereas relatively weakpre-TCR signals promote ab lineage commit-ment. This differed from a model proposingthat ab/gd lineage choice is determined beforeexpression of the gdTCR or the pre-TCR (Kangand Raulet 1997). The importance of TCR sig-nal strength in ab/gd lineage choice was testedin two studies that used an experimental systemwhere all immature DN thymocytes express asingle transgenic gdTCR (Haks et al. 2005;Hayes et al. 2005). Attenuation of gdTCR sig-naling potential by reducing the number of zITAMs (Hayes et al. 2005) or by changing theaffinity of the gdTCR-MHC interaction (Hakset al. 2005) resulted in a diversion from the gd

to the ab lineage supporting the signal strengthmodel of ab/gd lineage commitment. Con-sistent with these results, single cell lineagetracing experiments also demonstrated thatab/gd lineage choice does not take place priorto pre-TCR/gdTCR expression (Kreslavskyet al. 2008).





A large body of work derived from severalindependent laboratories supports the ideathat ITAM-mediated signal amplification playsa critical role in selection of the mature T-cellrepertoire. In DP thymocytes, productive rear-rangement of the TCRa locus results in surfaceexpression of clonally distinct abTCR com-plexes. To ensure that only those cells that ex-press TCRs with the appropriate specificities

are allowed to mature, DP thymocytes are sub-jected to a selection process on the basis of theirTCR specificity for peptideþMHC that pro-motes the survival and differentiation of func-tionally competent cells (positive selection)and triggers the deletion of overtly autoreactivecells (negative selection) (Sebzda et al. 1999;Starr et al. 2003). Positive selection is thoughtto be mediated by relatively weak non-agonist

Table 2. Roles of ITAMs within the CD31, CD3g, CD3d, and TCRz chains in T-cell development and function

Subunit Experimental approach Effects on development/function References

CD31 Reconstitution of CD312/2 micewith a transgene encoding amutant form of CD31containing a Y!F substitutionin the distal tyrosine residue ofthe CD31 ITAM

.Restores T-cell development toCD312/2 mice

Sommers et al. 2000

.Impaired proliferative responsesand cytokine responses whenlow doses of stimulatingantibody are used

.Defects in survival of T cellsexpressing TCRs with lowaffinity to self-ligands

CD3g Knock-in lacking the CD3g ITAM .Impaired efficiency of positivebut not negative selection

Haks et al. 2001;Haks et al. 2002

.Defects in proximal TCRsignaling events

.Proliferative responses andcytokine responses areequivalent to those ofwild-type mice

TCRz Reconstitution of TCRz2/2 micewith transgene encodingtruncated form of TCRzcontaining no ITAMs

.Restores T-cell development toTCRz2/2 mice

Shores et al. 1994;Love et al 2000;Ardouin et al.1999; Pitcheret al. 2003;Pitcher et al. 2005

.Defects in selection ofthymocytes expressing TCRswith low affinity to self-ligands

.Impaired proliferative responsesand cytokine responses whenlow concentrations of peptideare used

Reconstitution of TCRz2/2 micewith transgene encodingmutated form of TCRzcontaining Y!F substitutionsin all ITAMs.

.Restores T-cell development toTCRz2/2 mice

.Defects in selection ofthymocytes expressing TCRswith low affinity to self-ligands

All RAG12/2 mice reconstitutedwith CD3- and TCRz-deficientbone marrow transduced withretroviral vectors encodingITAM-mutant CD3 and TCRzchains

.Restoration of T-celldevelopment requires theexpression of at least 7 ITAMs

Holst et al. 2008

.Impaired proliferative responses

.Autoimmunity is observed whentwo to six ITAMs are expressed





peptides (Hogquist et al. 1994; Santori et al.2002) indicating that amplification of TCR sig-nals generated from such interactions may beespecially important for positive selection.Consistent with this notion, positive selectionwas found to be markedly impaired in z-deficient mice reconstituted with transgenesencoding ITAM mutant z chains (Shores et al.1994; Ardouin et al. 1999; Pitcher et al. 2005b).Moreover, in each of these model systems, theextent to which positive selection was compro-mised was directly related to the number of zITAMs that had been deleted or inactivated.Interestingly, analysis of positive selection usingdifferent abTCR transgenes demonstrated thenumber of TCR ITAMs that were required forpositive selection was dependent upon the pre-sumed affinity of the TCR for its selecting selfligand. DP thymocytes expressing TCRs thatare thought to bind to self peptideþMHCwith relatively high affinity could be positivelyselected even if several z ITAMs were deleted,whereas positive selection of thymocytes ex-pressing relatively low affinity TCRs requiredall 10 TCR ITAMs (Love et al. 2000). Thus, therequirement for ITAM-mediated signal ampli-fication was most clearly evident under con-ditions where positive selection was inducedby weak TCR-ligand interactions.

ITAM-MEDIATED SIGNAL AMPLIFICATIONAND CENTRAL TOLERANCE

The discovery that TCR ITAM multiplicitywas important for positive selection raised thequestion of whether negative selection is alsocompromised when the number of TCR ITAMsis reduced. The effect on negative selectionof mutating or removing one or more of the z

ITAMs was examined in two of the three exp-erimental models described above. In bothcases, negative selection was found to be com-promised when the number of z ITAMs wasreduced (Shores et al. 1997; Love et al. 2000;Pitcher et al. 2005a). Similar to the effects onpositive selection, the impact on negativeselection was directly related to the numberof ITAMs that were eliminated. Importantly,although potentially auto-reactive T cells were

generated in these mice, no overt signs ofautoimmune disease were observed. Theabsence of autoimmune disease in z ITAMmutant mice is consistent with the hypothesisthat the reactivity of T cells is “tuned” duringthymocyte selection through the regulatedexpression of compensatory molecules such asCD5 which function to modulate the integratedTCR signaling response (Grossman and Singer1996; Azzam et al. 2001; Wong et al. 2001;Saibil et al. 2003). However, a recent reporthas challenged this concept of thymocyte selec-tion (Holst et al. 2008). In this study, retrovirusencoded wild-type or ITAM mutant CD3g,2d, 21, and z chains (termed retrogenics)were used to reconstitute bone marrow cellsfrom CD312/2, z2/2, or CD312/2 x z2/2

mice. In these experiments, mice in whichT-cell development was reconstituted with con-structs that yielded fewer than seven functionalITAMs per TCR developed a lethal, multi-organautoimmune disease that was attributed to fail-ure of central tolerance (Table 2). The reason forthe striking discrepancy between these resultsand those of prior studies are presently unclearbut may relate to the different experimentalsystems employed. A potential drawback ofall three studies is that transgenic or retrogenicreconstitution does not replicate endogenousgene expression. Notwithstanding, these newresults, which imply that genetic or environ-mental changes that attenuate TCR signalingin preselection thymocytes can result in failureof central tolerance, call for additional studiesto resolve this important issue.

CONCLUDING REMARKS

Research over the past two decades since thediscovery and initial characterization of ITAMshas served to substantially advance our under-standing of the mechanisms by which ITAMsfunction to initiate signaling by the TCR. Cur-rently, the preponderance of data support theidea that the multiple TCR ITAMs functionmainly to amplify TCR signals and suggestthat this capacity for signal amplification isespecially critical for selection of the T-cell rep-ertoire. Data generated from experiments in





which the number of ITAMs per TCR arereduced have yielded conflicting results regard-ing the effects of these alterations on central tol-erance and the risk for autoimmune disease.Because these findings impact our basic under-standing of thymocyte selection, it will beboth important and worthwhile to establishmore physiologically appropriate models, suchas “knockin” mutations to resolve this issue.Experiments revisiting the question of whetherindividual TCR ITAMs selectively bind to pote-ntial downsteam effectors are also overdue andare especially promising as they may yield newinsights into the “raison d’etre” for the multipleITAM configuration of the TCR complex.

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published online April 28, 2010Cold Spring Harb Perspect Biol Paul E. Love and Sandra M. Hayes ITAM-mediated Signaling by the T-Cell Antigen Receptor

Subject Collection Immunoreceptor Signaling

Serine/Threonine KinasesThe Coordination of T-cell Function by

David Finlay and Doreen Cantrellof T Cell ActivationPerspectives for Computer Modeling in the Study

Altan-BonnetJesse Coward, Ronald N. Germain and Grégoire

ReceptorITAM-mediated Signaling by the T-Cell Antigen

Paul E. Love and Sandra M. Hayes Initiation of Signalinginto Receptor Assembly, Ligand Recognition, and Structural Biology of the T-cell Receptor: Insights

Call, et al.Kai W. Wucherpfennig, Etienne Gagnon, Melissa J.

Protein SLP-76Hematopoietic Cell Lineages by the Adaptor Coordination of Receptor Signaling in Multiple

Martha S. Jordan and Gary A. KoretzkySignaling Cross TalkInhibitory Pathways in Innate Immune Cells: Src-family and Syk Kinases in Activating and

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B-cell ActivationThe Cytoskeleton Coordinates the Early Events of

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B viaκAntigen Receptor Signaling to NF-

Pelzer, et al.Margot Thome, Jean Enno Charton, Christiane

MeshworksMediation of T-Cell Activation by Actin

Peter Beemiller and Matthew F. KrummelInitiation of B-Cell Receptor SignalingIt's All About Change: The Antigen-driven

Wanli Liu, Hae Won Sohn, Pavel Tolar, et al.

Kinase, ItkT-Cell Signaling Regulated by the Tec Family

Joseph, et al.Amy H. Andreotti, Pamela L. Schwartzberg, Raji E.

ZAP-70: An Essential Kinase in T-cell Signaling

Au-Yeung, et al.Haopeng Wang, Theresa A. Kadlecek, Byron B.

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FunctionLipid Signaling in T-Cell Development and

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S. ShawMichael L. Dustin, Arup K. Chakraborty and Andrey

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